Watching a virus grow
Watching a virus grow
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观察病毒的生长
DOI：
10.1073/pnas.1915986116
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发表时间：
2019
期刊：
影响因子：
--
通讯作者：
Dragnea, Bogdan
中科院分区：
文献类型：
--
作者：
Dragnea, Bogdan
迄今为止发现的最强大的分子马达之一属于噬菌体，它是一种感染细菌的病毒 (1)。该电机用于将坚硬的双链 DNA (dsDNA) 包装到称为原衣壳的预组装蛋白质多面体容器中。 DNA 比衣壳长约 50 倍，并被电机以接近晶体的密度包装和压缩。衣壳能够承受高达 40 个大气压的正压差 (2)。目前对这一令人着迷的生物过程的理解水平在很大程度上是由于适应噬菌体的包装动力学的单一方法的发展而成为可能的。然而，对一种病毒有效的方法可能很难适用于另一种病毒。因此，在大量单链 RNA (ssRNA) 病毒（地球上最大的病毒群）中，核酸包装与自发组装同时发生，而且实际上是自发组装的一部分。这里不涉及 ATP 驱动的电机。在平衡状态下，RNA 施加的最终压力不是很大的正压力，而是适度的负压力 (3)。解开两种类型的并发驱动相互作用（外壳蛋白之间的相互作用以及外壳蛋白与 RNA 之间的相互作用）之间的相互作用绝非易事。雪上加霜的是，中间体的存在时间非常短暂（毫秒），而可在体外组装的 ssRNA 病毒的尺寸却很小（数十纳米）。这就是为什么到目前为止，ssRNA病毒组装的主要特征主要由理论模型、静态数据的结构论证和整体平均动态实验提供。在单一方法中，能够提供与 20 年前应用于 dsDNA 噬菌体的方法相同水平的实时动态细节和直接洞察力的方法很少。在 PNAS 中，Garmann 等人 (4) 令人信服地证明了宽视场光学显微镜方法对 ssRNA 噬菌体 MS2 组装体进行严格控制的实时单粒子研究的潜力，具有接近分子的精度和广泛的时间动态范围。为了实时测量组装动力学，Garmann 等人 (4) 采用了一种称为干涉散射显微镜 (5) 的成像技术。在他们的方法中，稀疏分布的单分子病毒RNA通过柔性接头连接到溶液中显微镜盖玻片的表面。注射病毒外壳蛋白后，RNA−蛋白缔合导致束缚 RNA 位置的有效局部蛋白密度增加。由于蛋白质的光学偏振性与水的光学偏振性不同，因此不断生长的核蛋白复合物会增加光散射。散射光相对于入射光发生相移。这种相移提供了一种增强检测器平面对比度的方法，其中相干散射光与盖玻片反射的光发生干涉。因此，多个生长的病毒颗粒可以通过电荷耦合装置实时并行成像，作为强度与局部质量积累成比例的衍射极限点。报告的检测限为 1 Hz 带宽下 6 个外壳蛋白二聚体。
One of the most powerful molecular motors discovered, to date, belongs to a phage, a type of virus that infects bacteria (1). The motor serves to package stiff, double-stranded DNA (dsDNA) into a preassembled, proteinaceous polyhedral container called the procapsid. The DNA is about 50 times longer than the size of the capsid and gets packaged and compressed by the motor at near-crystalline densities. The capsid holds up its end of the bargain, withstanding positive pressure differentials of up to 40 atm (2). The level of current understanding of this fascinating biological process was made possible, in great measure, by the development of in singulo approaches to packaging dynamics, adapted to phages. However, what works for one virus may hardly apply to another. Thus, in a great number of single-stranded RNA (ssRNA) viruses—the largest virus group on Earth—nucleic acid packaging occurs concurrently with, and indeed is part of, spontaneous assembly. No ATP-powered motor is involved here. Instead of great positive pressures, the final pressure exerted by the RNA, at equilibrium, is modest and negative (3). Disentangling the interplay between the 2 types of concurrent driving interactions—those among coat proteins and those between coat proteins and RNA—is far from trivial. Adding to the challenge, intermediates have a fleeting existence (milliseconds), while the sizes of ssRNA viruses that can assemble in vitro are small (tens of nanometers). This is why, up to now, the principal features of ssRNA virus assembly have been mainly supplied by theoretical models, structural arguments from static data, and ensembleaveraged dynamic experiments. In singulo methods that would offer the same level of real-time dynamics detail and direct insight as those applied to dsDNA phages 2 decades ago have been scarce. In PNAS, Garmann et al.(4) provide a compelling demonstration of the potential held by a wide-field optical microscopy method for tightly controlled, real-time single-particle studies of the ssRNA bacteriophage MS2 assembly with near-molecular accuracy and broad temporal dynamic range. To measure assembly kinetics in real time, Garmann et al.(4) adapt an imaging technique called interferometric scattering microscopy (5). In their approach, sparsely distributed single molecules of viral RNA are tethered via flexible linkers to the surface of a microscope coverslip, in solution. Upon injection of virus coat proteins, RNA− protein association results in an increase of the effective local protein density at tethered RNA locations. Since the optical polarizability of proteins is different from that of water, there is increased light scattering by the growing nucleoprotein complex. The scattered light is phase-shifted with respect to the incident light. This phase shift provides a way to boost contrast at the detector plane, where the coherent scattered light is made to interfere with light reflected by the coverslip. Thus, multiple growing virus particles can be imaged in parallel via a charge-coupled device in real time, as diffractionlimited spots of an intensity proportional to the local mass accumulation. The reported detection limit is 6 coat-protein dimers at 1-Hz bandwidth.
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噬菌体物理学
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